PROJECT SUMMARY This proposal aims to elucidate the mechanisms and implications of step-wise oxidation of 5-methylcytosine by TET family enzymes. Modifications to cytosine bases play an important role in diverse processes, including embryonic development, pluripotency and oncogenesis. The best studied cytosine modification is methylation at the 5-position (5mC), which typically occurs at cytosine-guanine dinucleotides (CpGs) in the genome. While stable CpG methylation can mediate gene silencing in processes such as imprinting, dynamic changes in CpG methylation are also important in cellular differentiation and induced pluripotency. The recent discovery that 5mC can be oxidized by TET family enzymes has shed new light on the mechanisms involved in DNA methylation dynamics and has greatly increased the potential coding capacity of CpGs. There are three different TET family members that are differentially expressed and likely have both complementary and redundant roles. These enzymes are Fe(II)/?-ketoglutarate-dependent dioxygenases that can oxidize 5mC to generate 5-hydroxymethylcytosine (5hmC). Importantly, 5hmC is itself a substrate for further oxidation, generating 5-formylcytosine (5fC), and 5fC can be oxidized yet further to 5-carboxylcytosine (5caC). These three different oxidized 5mC bases (ox-mCs) are postulated to play two important biological roles: as intermediates in the process of DNA demethylation and as independent epigenetic marks. A critical goal in the field is understanding what distinct roles are played by each of the ox-mC bases. However, we currently lack an understanding of the mechanisms that regulate which modification is introduced at a given CpG by the different isozymes, nor can we dissociate these three intricately linked ox-mCs from one another. Our proposal aims to fill this gap by using a combination of innovative enzymatic assays and targeted active site manipulation. In order to track oxidation at a single site, we have developed isotopologue-based assays using specific radiolabeled or heavy-atom labeled 5mC. To inform how the extended epigenome is established and maintained, we will exploit our assays to determine the preferences for 5mC, 5hmC or 5fC as substrates and how opposite strand CpG modification influences each TET isozyme. To connect the biochemical preferences to patterns of activity in cells, we will pursue a cellular model that allows for expression of each TET isozyme in isolation and characterize the pattern of ox-mCs and their coupling on opposite strands. By manipulating the TET active site, we have also discovered remarkable mutants that are specifically deficient in later oxidation steps and now aim to decipher the mechanism at play in these ?5hmC-dominant? variants. We propose to exploit our discovery to determine whether 5hmC alone, or 5fC/5caC also, are required for reprogramming of fibroblasts to induced pluripotent stem cells. Thus, together our aims encompass mechanism and function: to elucidate how ox-mCs are generated and to dissect the distinguishing roles of 5hmC, 5fC and 5caC in important epigenetic processes.